The acetolactate synthase (ALS) enzyme, or acetohydroxyacid synthase (AHAS) enzyme, is an essential enzyme in branched-chain amino acid biosynthesis, and is the target site of five families of herbicides referred to as ALS inhibitors. Waterhemp (Amaranthus tuberculatus) is considered one of the most problematic weeds in the Midwest cropping region. The evolution of herbicide resistance and multiple resistance mechanisms within the species is one of the major properties making it difficult to control, and ALS-resistant waterhemp populations have been found and studied considerably. A waterhemp population (designated MCR) from Illinois with resistance to HPPD and atrazine was found to segregate for both high and moderate levels of resistance to ALS inhibitors. Plants in this population with high-level resistance had the Trp574Leu ALS mutation, which is present in other waterhemp populations resistant to ALS inhibitors. Plants from the MCR population that showed only moderate levels of resistance to ALS inhibitors did not have this mutation. Thus, research was conducted to investigate the resistance mechanism in the waterhemp plants with moderate resistance to ALS-inhibitors. Plants with moderate resistance were crossed and the resulting progeny where characterized. Firstly the ALS gene of the progeny was sequenced and in vitro ALS enzyme assays were conducted, and results indicated that the plants lacked a target-site mutation. Secondly, a series of greenhouse dose-response experiments were conducted to evaluate the resistance level across different chemical families of ALS-inhibitors. Thirdly, malathion, a cytochrome P450-inhibiting pesticide, was incorporated with ALS-inhibitor application to unveil the possible mechanism of resistance. Based on the results obtained, it was concluded that both target-site-mutation-based and metabolism-based ALS resistance mediated by cytochrome P450s exist in the original MCR population.